This article was supported by Award Number R01GM087227 from the National Institute of General Medical Sciences, the Norman Ackerman Advanced Research Program, and the Robert A. Welch foundation (Grant A-1769).

1. SPPS：CK（TMR）TATG（fTAT）合成注意：dfTAT产生于两个步骤：合成的单体fTAT通过固相肽合成接着二硫键二聚化以形成dfTAT的。 <ol><li>在一个标准的50 ml的SPPS容器溶胀二甲基甲酰胺（DMF）将500mg Rink酰胺MBHA树脂的1小时。 </li><li>通过温育的Fmoc保护的树脂中20％哌啶溶液（20％哌啶的DMF，将10毫升（0.30毫摩尔）进行Fmoc去保护。（1×5分钟和1×15分钟）用洗涤步骤使用贯彻去保护步骤两次DMF（洗涤步骤包括冲洗所述树脂多次使用的约150毫升DMF，总体积并除去通过真空过滤）中的溶剂中反应之间。 </li><li>合成CK（TMR）RKKRRQRRRG（fTAT）在Rink酰胺MBHA树脂。使用下面的Fmoc保护的氨基酸：Fmoc-赖氨酸（MTT）-OH（仅在N末端），将Fmoc-赖氨酸（BOC）-OH，使Fmoc-Gly-OH，Fmoc基 - 精氨酸（了Pbf）-OH，的Fmoc- GLN（TRT）-OH，和Fmoc-半胱氨酸（TRT）-OH。进行，使用N 2（20 PSI）以搅动该反应的反应。完成所有的反应在室温。 <ol><li>进行4小时的每个氨基酸偶联反应。对于每一个耦合，使用Fmoc基保护的氨基酸（1.2毫摩尔），N，N，N&#39;，N&#39; -四甲基-O-（1- -1H-苯并三唑 -1-基）脲六氟磷酸酯（HBTU）（0.44克，1.1毫摩尔），和二异丙基乙胺（DIEA）（0.51毫升，3.0毫摩尔）溶解在DMF中。以下的4小时耦合，用DMF洗涤树脂，并如在步骤1.2中所述进行Fmoc去保护步骤。 </li><li>重复，直到fTAT的线性肽链：合成（线性肽链CKRKKRRQRRRG没有TMR）。 </li><li>然后彻底使用第一DMF中，并以下，与二氯甲烷（DCM）洗涤树脂。冲洗树脂多次使用的约150毫升DMF或DCM的总体积与除去溶剂通过真空过滤。 </li><li> ü本身MALDI-TOF，确认获得的肽具有正确的分子量。 <ol><li>通过将1毫克矩阵到一个50微升的0.01％TFA的水溶液和50微升的乙腈组成的溶液使一个矩阵组合。 </li><li>以确认线性肽链的质量，使用α氰基-4-羟基肉桂酸。运行粗肽的分析型HPLC和从纯峰的顶部收集50微升。 </li><li>以制备样品到板在MALDI板上：添加8微升肽到2微升基质溶液和就位在MALDI板上的空气干燥或在37℃的热板。 注意：精氨酸是已知的氨基酸难以夫妇在SPPS。成功的偶合是通过执行一个Kaiser试验14（例如，茚三酮试验）确定。这种测试允许的游离胺可能留下的未偶联在树脂上的检测。万一蓝色被检测（指示游离胺的存在），耦合小号TEPS是重复的。 </li></ol></li></ol></li><li>为CK（-NH-TMR）TATG（fTAT），裂解MTT保护基赖氨酸的ε-氨基上的CK（-NH-MTT）TATG用的1％三氟乙酸（TFA）和2％三异丙基硅烷组成的溶液（TIS）的DCM。 <ol><li>作为除去MTT保护基将导致黄色的外观，孵育用20毫升上述溶液的树脂5分钟，重复上述操作直至没有黄色是观察。之间在洗净用DCM和DMF的树脂。此外，因为TIS是清除剂，将清除的MTT法，一旦无黄颜色出现在MTT法的存在下该溶液中。 </li><li>添加用1％TFA的DCM溶液（无TIS）的附加溶液的树脂，以确保没有更多的MTT是除去并将溶液保持清澈。 </li></ol></li><li>溶解的三个组成部分：Carboxytetramethylrhodamine（TMR），HBTU，和DIEA（4，3.9和10等价于相对于树脂（以mg）的量）的DMF并添加此混合物到树脂中，进行该反应O / N使用干燥的N 2，以提供搅拌。 </li><li>以下的Fmoc脱保护和氨基酸缀合，用DCM洗涤树脂，并让其干燥。用于从树脂上的肽的完全切割，添加含有92.5％TFA，2.5％的溶液H 2 O的，2.5％，TIS和2.5％乙二硫醇（EDT）（总体积= 4毫升）的肽基树脂3小时，在室温以实现全球去保护和切割从树脂。 <ol><li>由排出溶液从步骤1.6成40 ml的Et 2 O等的沉淀用冷的无水乙醚（Et 2 O等）的粗肽的产品，旋下来，在4℃和4000×g离心20 - 25分钟。重复此步骤，以允许用冷无水乙醚2 O沉淀物的洗涤</li><li>重悬沉淀物在水（5ml或直至沉淀完全溶解），并冷冻干燥。然后重悬在0.1％TFA水溶液/乙腈制得的产品。 </li></ol></li> &lt;李&gt;用分析C18柱（5微米，4×150毫米），以分析每一个肽进行HPLC分析。使用1毫升/分钟的流速，并检测在214nm和550nm处。 </li><li>在C18 10×250毫米柱的肽纯化进行半制备型HPLC。使用的4毫升/分钟的流速，并检测在214nm和550nm处。对于所有的运行时，使用0.1％TFA水溶液（溶剂A）和90％乙腈，9.9％水和0.1％TFA（溶剂B）中的线性梯度。 </li><li> fTAT，预期质量：：2,041.17，观察到的质量：2040.66根据制造商的协议通过MALDI-TOF确定的肽的正确身份。 DEAC-K9，预期质量：1,412.97，观察肿块：1,415.59。使用α-氰基-4-羟基肉桂酸矩阵的MALDI-TOF。 </li></ol> 2.氧化反应：dfTAT代<ol><li>通气的磷酸盐缓冲盐水（PBS）pH 7.4的1小时（至少5毫升下面的反应）。 </li><li>在AER溶解fTAT（0.3毫克，1.5×10 -4毫摩尔）ated pH 7.4的PBS（5毫升）中。确保的pH为7.0之间 - 7.5在加入肽后。如果不添加氢氧化钠（1M，以小的增量1 - 5微升）以使pH恢复到7.4。 </li><li>注意：氧溶解在PBS作用以氧化硫醇基上fTAT和形成二硫键。 </li><li>章动反应O / N，以允许它直到完成（100％收率基于HPLC分析）反应。使用反相HPLC提纯产物和通过质谱法（MALDI-TOF）如在步骤1.9分析。预期质量：4,080.34，观察肿块：4,084.21。 </li><li>冷冻干燥纯dfTAT（0.71×10 -4毫摩尔），并悬浮在200μl水（浓度纯dfTAT = 356.3μM）。 </li></ol> 3.测量dfTAT浓度<ol><li>在149微升的50mM TCEP溶液重悬纯化dfTAT（通常为1微升取决于肽的纯化的量）的等分试样。 </li><li>注：在这一步dfTAT减记至莫nomer对口fTAT消除时出现的吸光度淬火由于TMR荧光团在dfTAT近距离（TMR在dfTAT消光系数ε减小相比TMR的降低fTAT的ε）。 </li><li>允许样品大约20分钟反应（分析性HPLC可用于确认形成fTAT的）。 </li><li>添加所有的溶液到石英比色皿，测量吸光度556纳米。 </li><li>使用比尔定律（A =εcl：ε= 91500 M-1 -1）来计算fTAT的浓度，确定dfTAT的浓度，并划分[fTAT]由两个。 </li></ol> 4.蜂窝交货实验<ol><li>种子在培养皿中的细胞（HeLa细胞，HDF， 等）在80汇合- 90％（例如，8孔或24孔皿）。生长的细胞在适当的培养基（例如，DMEM补充有10％FBS和青霉素/链霉素），直到80 -在37℃90％汇合湿润气氛含有5％ 的 CO 2。 </li><li> （通过添加200微升PBS，然后除去它三次）洗涤细胞，用PBS三次（3倍）。 </li><li>使dfTAT的5μM的工作浓度在NRL-15培养基稀释dfTAT（在水中）的股票（对于一个8井菜的总体积应为200微升）。 5微米dfTAT甲浓度导致有效递送在测试日期（ 图3）大多数细胞类型（在90％以上的细胞中存在的碟形高水平胞质递送）。然而，较低或较高的浓度可能是更充分的用于与高或低的倾向为渗透的细胞类型。 注意有关介质：NRL-15用于dfTAT的交付，因为它不含有半胱氨酸可能降低dfTAT二硫键。然而，我们的数据表明，这两个定期L-15（半胱氨酸）和DMEM可以用作传递介质。 L-15的包含还原氨基酸半胱氨酸但半胱氨酸presumabLY氧化在媒体上形成胱氨酸（DMEM是制定与胱氨酸）。因此dfTAT丝毫无损这些媒体和传送工作在相同的效率，与nrL15获得。 </li><li>孵育细胞dfTAT（5μM）具有或不具有货物（例如，绿色荧光蛋白（10μM）），并保持在37℃下进行1小时（温育时间可以减少，但dfTAT通常需要大约30至45分钟，以诱导内体泄漏）。 </li><li>用肝素（1毫克/毫升）中的L-15洗涤细胞（3次洗涤，建议）以除去dfTAT结合于细胞的质膜上。 </li><li>孵育细胞与细胞不透核染色剂，例如，SYTOX蓝，SYTOX绿（2μM在NRL-15），以确定细胞的质膜是否受到损害，根据（死细胞将同时活细胞不会被染色）制造商的协议。 </li><li>使用荧光显微镜（100X油浸或20X物镜）图像的细胞。使用RFP滤波器（图片dfTAT防爆=560±20纳米/ EM = 630±35纳米）。 注意：成功递送导致dfTAT的漫荧光整个细胞（评估蛋白质或感兴趣的肽的递送将取决于应用）。核仁由fTAT（dfTAT的时胞质条目的还原产物）的染色可以用作一个指示检测到的荧光是细胞内的。 fTAT将在几个小时内降解。在这一点上，降解片段的荧光将显示为点状。这不应该混淆为点状分布也可以看出，如果dfTAT仍然不成功内内体困（这可能发生，如果dfTAT出现在太低的浓度）。 </li></ol> MOI 5. Controling集中交付<ol><li>确定实现dfTAT中使用的细胞类型高效胞浆释放所需要的“最优配送”的浓度。在细胞培养与increasin执行荧光显微镜克浓度dfTAT的。最佳dfTAT浓度被定义为最小浓度，导致清除胞质“扩散”TMR荧光细胞的大约100％在培养。 </li><li>改变MOI的浓度用在共温育协议，同时保持dfTAT恒定的浓度（例如，使用dfTAT的最佳递送浓度）。改变培养时间以不足1小时也可以是一种选择，以改变的量的MOI递送。 </li></ol>

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The authors have nothing to disclose.

因为这用于dfTAT交付不应过分融合的细胞（&gt; 90％汇合）可能会影响传递效率。该细胞也应该是健康的：死细胞的培养可以释放凋亡片段与dfTAT可交互（例如，从DNA降解核）。这反过来又可以与递送效率和成像质量干扰。细胞应彻底洗净加入dfTAT之前删除FBS。 BSA存在的FBS可以结合dfTAT并且这可以降低dfTAT的递送效率。洗涤然而，应当小心以过大的力进行可能导致贴壁细胞从培养皿分离?...

递送蛋白质，肽或细胞不可渗透的小分子进入活细胞是在许多生物或生物技术应用（细胞成像，功能测定法，细胞重新编程等 ）1-4常常合乎需要的。许多递送方法已被报道，包括显微注射，电穿孔，或用载体试剂（例如，细胞穿透肽，例如TAT，脂质）5-7。每种技术一般都有特定的优点和缺点，可能使这些方法足以满足一定的应用，但不是为别人。常见问题涉及在少数细胞而不在整个贫困人口递送效率和/或控制的多少材料输送8,9，毒性或有害生理影响10,11，缺乏临时控制的，交付的缺乏（例如，显微注射）12，和复杂的化学缀合或制剂方案11。 S =“jove_content”&gt;最近，我们已经开发了一种新的交付策略绕开这些限制。这一战略依赖于一个名为dfTAT肽（二聚体荧光TAT）13。 dfTAT从公知的细胞穿透肽（CPP）的TAT而得。 dfTAT包含TAT的两个二硫键结合拷贝标记荧光团四甲基罗丹明。尽管他们的相似之处，TAT和dfTAT活性显著不同。 TAT是典型的内吞作用内化进入细胞。这CPP仍然但是在大多数被困在内涵体（这通常会导致细胞内的肽的点状分布时，利用荧光显微镜检查）。像TAT，dfTAT有效地被细胞内吞。然而，dfTAT不会留在里面内涵体困。相反，它介导内体泄漏的方式，是非常有效的。 dfTAT的内体裂解活性可以然后被利用通过一个简单孵育测定递送大分子。 ntent“&gt;传送过程的当前理解如下。dfTAT诱导巨吞。其结果是，细胞孵育dfTAT占用的可溶性蛋白，肽或小分子（感兴趣的分子，MOI）存在于介质通过流体相内吞作用（ 参见图1）。dfTAT和MOI之间的相互作用不是必须的，只要内吞途径中一起两个实体通信。正如dfTAT到达内吞细胞器的内腔中一个特定的阈值，它表示它的内体泄漏活性（的分子细节仍有待充分表征的）。漏细胞器的内腔的内容，因此，教学语言，然后被释放到细胞中。因此，这种方法是与MOI没有缀合或制剂计划必须很方便的。此外，由于dfTAT是不直接修改一个MOI，它还应不同的MOI功能干扰一旦细胞内递送的实现。此外，浓度dfTAT用于交付是独立于MOI的媒体。例如，dfTAT浓度可以保持不同的实验之间保持恒定，以保证在内体泄漏重现的效率。与此相反，MOI在媒体浓度可逐渐改变以达到所需水平的细胞质MOI交付。 实现了与dfTAT高内体泄漏效率显着地无害的许多测试日期的细胞。这是一个惊喜，因为内吞细胞器是细胞的重要组成部分，人们所期望的显着泄漏通过dfTAT介会伴随着有害的细胞反应。然而，处理的细胞增殖以相同的速率作为未经处理的细胞，并且不显示在其转录任何显著改变。此外，可交付使用可重复的递送效率分钟内反复，这表明细胞可以容忍或恢复从分娩过程，而不会丢失他们的能力，内吞作用或内体泄漏。 dfTAT交付和什么这些反应可能是有待探索的分子细节中微妙的细胞反应可能发生。然而，通过结合高效率，便利性的协议，并没有毒性，这种交付方法应该证明在许多细胞为基础的应用可以立即投入使用。本文提出的协议的目的是使该技术访问研究界。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Heparin&#160;</td>
			<td>Sigma</td>
			<td>CAS 9041-08-1</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX Blue</td>
			<td>Invitrogen</td>
			<td>S11348</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX Green</td>
			<td>Invitrogen</td>
			<td>S7020</td>
			<td></td>
		</tr>
		<tr>
			<td>nrL15 L-15 (&#8211;) cysteine</td>
			<td>Hyclone</td>
			<td></td>
			<td>Special order</td>
		</tr>
		<tr>
			<td>Fmoc-protected Amino acids</td>
			<td>Novabiochem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>dfTAT</td>
			<td></td>
			<td></td>
			<td>Samples will be provided upon request (contact: pellois@tamu.edu)</td>
		</tr>
		<tr>
			<td>Biosafety Cabinet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted epifluorescence microscope</td>
			<td>Olympus</td>
			<td>model IXB1</td>
			<td>equipped with a heating stage maintained at 37 &#176;C and with a Rolera-MGI Plus back-illuminated electron-multiplying charge-coupled device (EMCCD) camera (Qimaging).&#160;</td>
		</tr>
		<tr>
			<td>37 &#176;C humidified, 5% CO2 incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Peptide synthesizer or vessel to preform manual peptide synthesis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

delivery of proteins, peptides or cell-impermeable small molecules into live cells by incubation with the endosomolytic reagent dftat

大分子递送策略通常利用内吞途径蜂窝条目的路由。然而，内体截留严重限制与大分子穿透细胞的胞质空间的效率。最近，我们通过确定试剂dfTAT，标记有荧光团四甲肽TAT的二硫键二聚体规避这个问题。我们已经生成原型细胞穿透肽（CPP）的TAT，dfTAT，穿透活细胞和到达细胞具有特别高的效率的胞质空间的荧光标记的二聚体。胞质递送dfTAT的是在多种细胞系，包括原代细胞来​​实现的。此外，交货不会明显影响细胞的活力，增殖或基因表达。 dfTAT可以提供小分子，肽，抗体，生物活性酶和转录因子。在这份报告中，我们描述参与DFTA协议T合成和细胞输送。手稿描述如何通过改变蛋白质的细胞外给药量以控制蛋白质递送至细胞的胞质空间的量。最后，这种新技术和涉及验证输送步骤的当前局限性进行了讨论。所描述的方案应为基于细胞的测定以及对于离体操作的细胞和重编程是非常有用的。

为了评估fTAT和dfTAT之间的差，HeLa细胞被温育1小时以每种肽，以确定在他们的细胞定位的差异。使用荧光显微镜的两个CPP的内化进行了评估。 图2表明，fTAT（20μM）定位于点状分布。这种分布是一致的肽剩余的夹带里面内涵体。与此相反，dfTAT（5微米）的荧光信号显示整个胞质溶胶和细胞核的均匀分布;图3示出了 dfTAT的胞质分布在许多不同的细胞系，包括COLO 31...

Watch this Scientific Journal Video about Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT at JoVE.com

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky.

交货蛋白质，多肽或细胞不可渗透的小分子进入活细胞被孵化与核内体溶解试剂dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

delivery-proteins-peptides-or-cell-impermeable-small-molecules-into

Research

JoVE Journal

Bioengineering

10.2K 次观看.  Texas A&M University. We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky. 

视频: Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

无试剂检测核酸，蛋白质和小分子的电化学DNA生物传感器的制备

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

科研

生物工程

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

活细胞成像的移植细胞在一个三维矩阵表示荧光标记的蛋白质

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5&#8217; and 3&#8217; ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

单个RNA分子的光镊纳米操作

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

血浆或尿液的水凝胶纳米粒子收获检测低丰度蛋白

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs through the blood-brain barrier (BBB), causing difficulties for effective pharmacological therapies. In recent years, different strategies for promoting brain targeting have aimed to improve drug delivery and activity at this site, including innovative nanosystems for drug delivery across the BBB. In this context, an in vitro approach based on a simplified cellular model of the BBB provides a useful tool to investigate the effect of nanoformulations on the trans-BBB permeation of molecules. This study describes the development of a double-layer BBB, consisting of co-cultured commercially available primary rat brain microvascular endothelial cells and astrocytes. A multiparametric approach for the validation of the model, based on the measurement of the transendothelial electrical resistance and the apparent permeability of a high molecular weight dextran, is also described. As proof of concept for the employment of this BBB model to study the effect of different nanoformulations on the translocation of fluorescent molecules across the barrier, we describe the use of fluorescein isothiocyanate (FITC), loaded into ferritin nanoparticles. The ability of ferritins to improve the trans-BBB permeation of FITC was demonstrated by flux measurements and confocal microscopy analyses. The results suggest this is a useful system for validating nanosystems for delivery of drugs across the BBB.

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

A method to establish an in vitro model of blood-brain barrier based on a co-culture of rat brain microvascular endothelial cells and astrocytes is described and validated. This system proved to be a valid tool to study the effect of nanoformulation on the trans-barrier permeation of fluorescent molecules.

跨越大鼠血脑屏障FITC加载铁蛋白体外渗透：一个模型来研究Nanoformulated分子的交付

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

生物活性蛋白或多肽在水凝胶上的应用

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior in such restrictive conditions. To address this issue, we have developed growth chambers for tip-growing plant cells that contain a series of narrow, micro-fabricated gaps (~1 &#181;m) in a poly-dimethylsiloxane (PDMS) substrate. This transparent material allows the user to monitor tip elongation processes in individual cells during microgap penetration by time-lapse imaging. Using this experimental platform, we observed morphological changes in pollen tubes as they penetrated the microgap. We captured the dynamic changes in the shape of a fluorescently labeled vegetative nucleus and sperm cells in a pollen tube during this process. Furthermore, we demonstrated the capability of root hairs and moss protonemata to penetrate the 1 &#181;m gap. This in vitro platform can be used to study how individual cells respond to physically constrained spaces and may provide insights into tip-growth mechanisms.

We describe a method to investigate the capability of tip-growing plant cells, including pollen tubes, root hairs, and moss protonemata, to elongate through extremely narrow gaps (~1 &#181;m) in a microfluidic device.

微流控装置在极小空间中研究尖端生长植物细胞的伸长能力

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior ...

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all variations are based on the 20 canonical amino acids (cAAs). As a way to introduce additional physicochemical properties into polypeptides, the incorporation of non-canonical amino acids (ncAAs) is increasingly used in protein engineering. Due to their relatively short length, the modification of ribosomally synthesized and post-translationally modified peptides by ncAAs is particularly attractive. New functionalities and chemical handles can be generated by specific modifications of individual residues. The selective pressure incorporation (SPI) method utilizes auxotrophic host strains that are deprived of an essential amino acid in chemically defined growth media. Several structurally and chemically similar amino acid analogs can then be activated by the corresponding aminoacyl-tRNA synthetase and provide residue-specific cAA(s) &#8594; ncAA(s) substitutions in the target peptide or protein sequence. Although, in the context of the SPI method, ncAAs are also incorporated into the host proteome during the phase of recombinant gene expression, the majority of the cell&#39;s resources are assigned to the expression of the target gene. This enables efficient residue-specific incorporation of ncAAs often accompanied with high amounts of modified target. The presented work describes the in vivo incorporation of six proline analogs into the antimicrobial peptide nisin, a lantibiotic naturally produced by Lactococcus lactis. Antimicrobial properties of nisin can be changed and further expanded during its fermentation and expression in auxotrophic Escherichia coli strains in defined growth media. Thereby, the effects of residue-specific replacement of cAAs with ncAAs can deliver changes in antimicrobial activity and specificity. Antimicrobial activity assays and fluorescence microscopy are used to test the new nisin variants for growth inhibition of a Gram-positive Lactococcus lactis indicator strain. Mass spectroscopy is used to confirm ncAA incorporation in bioactive nisin variants.

The protocol presents the Escherichia coli-based selective pressure incorporation of non-canonical amino acids (ncAAs) into the lactococcal antimicrobial peptide nisin. Its properties can be changed during recombinant expression via substitution with desired ncAAs in defined growth media. Resulting changes in bioactivity are mapped by growth inhibition assays and fluorescence microscopy.

非规范氨基酸选择性压力合成抗菌肽的研究

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all ...

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different infectious diseases. In this article we demonstrate a method to produce a SAPN core containing a six-helix bundle (SHB) assembly that is capable of presenting antigens in a trimeric conformation. We describe the expression of the SHB-SAPN in an E. coli system, as well as the necessary protein purification steps. We included an isopropanol wash step to reduce the residual bacterial lipopolysaccharide. As an indication of the protein identity and purity, the protein reacted with known monoclonal antibodies in Western blot analyses. After refolding, the size of the particles fell in the expected range (20 to 100 nm), which was confirmed by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. The methodology described here is optimized for the SHB-SAPN, however, with only slight modifications it can be applied to other SAPN constructs. This method is also easily transferable to large scale production for GMP manufacturing for human vaccines.

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

大肠杆菌-表达自组装蛋白纳米颗粒的生产，用于需要三聚体异体的疫苗

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...